Possible relationships between tumorigenicity and expression of beta-GalNAcT-1, beta-GalNAcT-2, and alpha-GalNAcT-3 involved in Gg-\and Gb-core-containing glycosphingolipid biosynthesis and SAT-3 in LM1 biosynthesis will be a major emphasis of this research proposal. The overall immediate objectives of this research program are to solubilize and purify these three N-acetylgalactosaminyltransferases from guinea pig tumor cells (104Cl and 106B) and embryonic chicken brains by anion exchange and specific affinity chromatography. HPLC of 125I-GSLs (from cell surfaces)and enzymatic studies will also be carried out to compare the synthesis of Forssman antigen (glycolipid) during the growth cycle of guinea pig 104Cl and 106B cultured cells. Radioactive products will be isolated from [14C-Ac]-GbOse4Cer and [3H]GbOse3Cer after reaction with unlabeled UDP-GalNAc. The exact chemical linkages of the terminal GalNAc residues will be determined after permethylation of purified enzymatic products and characterization of the partially methylated radioactive [3H]galactose and [14C-Ac]GalNAc units obtained by autoradiography. Anomeric linkages will be determined by using highly purified beta-hexosaminidase (jack bean and papaya) and alpha-hexosaminidase (clam). Purification of alpha-hexosaminidase from clam is also part of this project. Differential inhibition of these three GalNAct activities will be studied in the presence of UDP, IDP, pCMB, and NEM. Biosynthesis in vitro of LM1 (sialosyl-neolactotetraosylceramide; NeuAc-nLcOse4Cer) in mouse N-18 clone and in human neuroblastoma 1MR-32 cells will be investigated in the third year of proposed research. Binding of [125I]-labeled GSLs, lectin (Eunonymus europeus) and toxins (cholera and ricin) to neuroblastoma cell surfaces will also be studied before and after chemically induced differentiation with (But)2 cAMP and HMBA. (A)